Antigen discovery has focused on using human serum samples collected from individuals that are naturally exposed to malaria and appear to develop effective immunity that prevents gametocytemia or blocks parasite transmission to mosquitoes, as tools to identify candidate vaccine antigens. Specifically, serum samples that have activity of interest are compared to sera that lack this activity, for their ability to select or recognize individual recombinant proteins constructs in P. falciparum expression libraries. Recombinant proteins identified through differential screening are then prepared as immunogens and tested for their ability to induce effective anti-gametocyte or transmission-blocking antibodies. 1. Phage Display Subtractive Screening was performed, using field sera having no transmission-blocking activity, for negative panning followed by positive panning with four different sera, each with natural transmission-blocking activity. Using this approach, sixteen different TBV candidates were recognized by at least two different sera with natural transmission-blocking activity. Of these, the top three TBV candidates were recognized by all four positive sera. 2. Genes corresponding to the top nine candidates were synthesized and cloned into mammalian expression vectors for DNA immunization of mice to examine their transmission-blocking activity by standard membrane feeding assay (SMFA). 3. Sera and purified IgG from mice immunized with the top candidate showed transmission-blocking activity comparable to the positive control Pfs25. 4. To examine these candidates further, the top three candidates recognized by all four positive sera were expressed in E. coli. 5. In FY 2017, the E. coli expressed and purified proteins were used to immunize mice either individually or in combination with either Pfs25 or Pfs230. The results showed that the positive controls Pfs25 and Pfs230 showed close to 100% TRA, but unlike what was observed for the Gene Gun study, the partial TRA activity of the top candidate was not reproducible. 6. The top three candidates were also examined by mice immunization by DNA electroporation which is known to induce higher immune response. Here, the positive control Pfs25 DNA vaccine also showed close to 100% TRA, but unlike what was observed for the Gene Gun study, the partial TRA activity of the top candidate was not reproducible.